Summary

During kidney morphogenesis, the formation of nephrons begins when
mesenchymal nephron progenitor cells aggregate and transform into epithelial
vesicles that elongate and assume an S-shape. Cells in different regions of
the S-shaped body subsequently differentiate into the morphologically and
functionally distinct segments of the mature nephron. Here, we have used an
allelic series of mutations to determine the role of the secreted signaling
molecule FGF8 in nephrogenesis. In the absence of FGF8 signaling, nephron
formation is initiated, but the nascent nephrons do not express Wnt4
or Lim1, and nephrogenesis does not progress to the S-shaped body
stage. Furthermore, the nephron progenitor cells that reside in the peripheral
zone, the outermost region of the developing kidney, are progressively lost.
When FGF8 signaling is severely reduced rather than eliminated, mesenchymal
cells differentiate into S-shaped bodies. However, the cells within these
structures that normally differentiate into the tubular segments of the mature
nephron undergo apoptosis, resulting in the formation of kidneys with severely
truncated nephrons consisting of renal corpuscles connected to collecting
ducts by an abnormally short tubular segment. Thus, unlike other FGF family
members, which regulate growth and branching morphogenesis of the collecting
duct system, Fgf8 encodes a factor essential for gene regulation and
cell survival at distinct steps in nephrogenesis.

Introduction

The mature nephron, the functional unit of the kidney (metanephros), is a
complex structure comprising a renal corpuscle, where blood is filtered, and a
tubular segment, where solutes and fluids required for homeostasis are
reabsorbed (see Fig. 1A).
Nephron formation is initiated by signals released from the tips of the
nascent branches of the ureteric bud-derived collecting duct system
(Saxen, 1987). These signals
induce cells in the metanephric mesenchyme to condense into pre-tubular
aggregates that develop into epithelial renal vesicles, which subsequently
elongate and develop into S-shaped bodies (see
Fig. 1A). Cells in the proximal
domain of the S-shaped body differentiate into specialized epithelial cell
types of the mature renal corpuscle, i.e. podocytes and Bowman's capsule
cells. The more distal domain of the S-shaped body differentiates into the
tubular portion of the nephron, which is segmented into proximal tubular, loop
of Henle, and distal tubular domains (see
Fig. 1A). The final stages of
nephron maturation are characterized by extensive elongation of the tubular
nephron segments and the expression of specialized nephron segment-specific
transport proteins (Nakai et al.,
2003).

The process of nephron induction and maturation is continually occurring in
the cortical region of the developing kidney, which is found progressively
further away from the original site of kidney induction as the organ grows.
Nephrogenesis is dependent on the maintenance of a nephron progenitor cell
population that is able to respond to the signals for nephron formation from
the collecting duct branch tips. These nephron progenitors are thought to
reside in the outermost region (`peripheral zone' in
Fig. 1A) of the expanding
kidney, which also contains cells that give rise to the stroma
(Hatini et al., 1996).

At present, little is known about the molecules that control the early
steps in nephrogenesis. Genetic analysis in mice has identified WNT9b,
released from the developing collecting ducts, as a signaling molecule that
induces nephrogenesis (T. Carroll and A. McMahon, personal communication).
Another WNT family member, WNT4, produced by nephron progenitors within the
pre-tubular aggregate, is thought to be required for the mesenchymal to
epithelial transition that leads to the formation of renal vesicles
(Stark et al., 1994;
Kispert et al., 1998), whereas
the transcription factor LIM1 (LHX1 – Mouse Genome Informatics) is
necessary for the progression of renal vesicles to the S-shaped body stage
(Kobayashi et al., 2005).
Presenilins, which are necessary for the proteolytic activation of Notch,
appear to function at a slightly later stage in nephrogenesis, as S-shaped
bodies develop in the absence of Psen1 and Psen2, but the
nephrons that form are severely truncated, and lack renal corpuscles and
proximal tubules (Cheng et al.,
2003; Wang et al.,
2003). Similarly, embryos that lack Brn1 (Pou3f3–
Mouse Genome Informatics), a POU-domain transcription factor, initiate
nephrogenesis but form truncated nephrons that lack much of the loop of Henle
(Nakai et al., 2003).

Members of the Fibroblast Growth Factor (FGF) family of secreted signaling
molecules are also known to play a role in kidney morphogenesis, but thus far
genetic analyses have revealed only an indirect role for FGF signaling in
nephron formation. Thus, in the absence of either FGF7 or FGF10
(Qiao et al., 1999;
Ohuchi et al., 2000), or the
receptor they activate (FGFR2-IIIb)
(Revest et al., 2001;
Zhao et al., 2004), the number
of collecting duct branches is reduced. This in turn results in a decrease in
the number of nephrons that form. Another FGF family member, Fgf8, is
expressed in the developing kidney
(Crossley and Martin, 1995;
Mahmood et al., 1995), but its
function in kidney development has not been assessed. In this study, we have
used an Fgf8 conditional-null allele
(Meyers et al., 1998) to
demonstrate that FGF8 is essential for the earliest steps in nephrogenesis,
and an Fgf8 hypomorphic allele
(Meyers et al., 1998) to show
that the survival of cells that develop into the tubular segment of the
nephron is dependent on FGF8.

Materials and methods

Mouse mutants

All mouse lines were maintained on mixed genetic backgrounds. Two different
null alleles, Fgf8Δ2,3
(Meyers et al., 1998) and
Fgf8lacZ (D. Brown and G.R.M., unpublished), were used
interchangeably in this study; both are referred to as
Fgf8null. Embryos were genotyped for Fgf8 alleles
and for the presence of Cre as previously described
(Sun et al., 1999;
Sun et al., 2002), and for
Fgf8lacZ by staining the tissue remaining after
metanephros removal for β-galactosidase activity. Wild-type metanephroi
for analysis of Fgf8, Wnt4 and FGF receptor gene expression were
isolated from outbred mice (CD1, Charles River). To stage embryos, noon of the
day when a vaginal plug was detected was considered to be embryonic day (E)
0.5.

Quantification of Fgf8 RNA

Both metanephroi from each embryo analyzed were homogenized in DEPC-treated
phosphate-buffered saline (PBS) with 0.1% Triton X-100 and total RNA was
extracted using TRIzol Reagent (Invitrogen 15596-026). Following isopropanol
precipitation and resuspension in 15 μl water, reverse transcription was
performed using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT;
Invitrogen 28025-013) to synthesize cDNA. Quantitative PCR was performed using
an ABI Prism 7900 HT Sequence Detection System. The primers used to amplify
Fgf8 sequences spanned exons 2 and 3 (forward primer,
5′-TCTCCAGCACGATCTCTGTGAA-3′; reverse primer,
5′-GGAAGCTAATTGCCAAGAGCAA-3′). The TaqMan® probe
sequence was 5′-ACGCAGTCCTTGCCT-3′. Gapdh sequences were
amplified and the amount of product was used for normalizing samples.

In situ hybridization

RNA in situ hybridization on whole mounts or on 10 μm cryosections were
performed according to standard protocols. For analysis of vibratome sections,
kidneys were fixed in 4% paraformaldehyde (PFA), embedded in 4% low-melting
point agarose in PBS and sectioned at 100 μm. For each gene analyzed, all
sections collected from a single kidney were processed together using a
whole-mount in situ hybridization protocol. The images presented here show a
representative section from each kidney. To generate digoxigenin-labeled
probes, we used plasmids containing mouse Fgf8, Wt1, Gdnf, Foxd1,
Lim1 and Umod sequences for in vitro transcription, whereas
probes for Slc34a1, Slc12a3, Wnt4 and Podxl were transcribed
using PCR products as templates (see Table S1 in the supplementary
material).

X-Gal staining to detect lacZ activity

Prior to X-Gal staining, tissues were embedded in a 1:1 mix of 20% sucrose
and OCT (Tissue-Tek 4583), and cryosectioned at 10 μm. Sections were then
fixed in 0.5% PFA for 10 minutes on ice, rinsed three times for 10 minutes
each in PBS, stained overnight at 37°C using standard X-gal staining
protocols, then counterstained with Eosin.

Detection of cell death

LysoTracker (Molecular Probes L-7528) labels acidic compartments within
apoptotic cells themselves, as well as in healthy cells that are engulfing
apoptotic debris (Zucker et al.,
1999; Schaefer et al.,
2004). For LysoTracker analysis, embryonic kidneys were isolated
in Hank's balanced salt solution (HBSS), incubated with 5 μl/ml LysoTracker
solution in HBSS at 37°C for 45 minutes, rinsed in HBSS, fixed in 4% PFA
in PBS and stored at –20°C in 100% methanol. Following rehydration
into PBS, LysoTracker-labeled kidneys were vibratome sectioned and processed
for anti-PAX2 immunofluorescence. Images were photographed using either a
stereomicroscope or a confocal microscope. For TUNEL analysis, embryonic
kidneys were fixed in 4% PFA. After overnight incubation in 20% sucrose,
kidneys were mounted in OCT and cryosectioned. Apoptotic cell death was
detected with the In Situ Cell Death Detection Kit, TMR red, following
manufacturer's instructions (2156792 Roche). When required, immunofluorescence
assays were performed after TUNEL analysis.

Embryonic kidney explant cultures

Kidney rudiments were isolated from ∼E11.5 mouse embryos. For
experiments involving isolated metanephric mesenchyme (MM), the ureteric bud
(at no later than the early T-stage) was removed from the kidney rudiment
using fine tungsten needles, following treatment with collagenase and DNAse A,
as previously described (Qiao et al.,
1995). Intact kidney rudiments and isolated MMs were cultured in
Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum on
polycarbonate membrane filters (0.4 μm pore size,
Transwell®, Corning). For experiments on the effects of
anti-FGF8 antibody, either normal goat serum (control) or blocking antibody
against FGF8 (10 μg/ml, R&D Systems, AF423NA) was added to the culture
medium. For experiments with MMs, dorsal spinal cord was added to the
cultures. After 2 or 3 days, the tissues were fixed in 4% PFA and processed
for immunofluorescence analysis in whole mount.

Isolation of nephrons

Kidneys were isolated from E18.5 mouse embryos, fixed in 4% PFA and
vibratome sectioned at 200 μm. Sections were incubated in 0.2% collagenase
(from Clostridium Histolyticum) with 50 units/ml DNase I for 30
minutes at 37°C, rinsed in PBS and further digested in concentrated HCl
for 15 minutes at 37°C. After extensive washing, nephrons were dissected
in cold PBS under a stereomicroscope using fine tungsten needles.

Results

Fgf8 is expressed in the developing nephron

The earliest stage at which we detected Fgf8 RNA in the kidney
primordium was ∼E12 [the 48-somite stage (som)], when it was observed in a
continuous domain immediately surrounding the emerging ureteric bud
(Fig. 1B,C, and data not
shown). By ∼E12.5 (52 som), Fgf8 expression appeared to have
resolved into numerous discrete spots near the periphery of the kidney
primordium (Fig. 1D). This
punctate staining pattern was observed until at least E18.5
(Fig. 1E, and data not shown).
Analysis of sections at E16.5, which contain nephrons at all stages in their
development, revealed that the punctate staining in whole mounts reflected
abundant Fgf8 RNA in pre-tubular aggregates and early renal vesicles
(Fig. 1E′, and data not
shown). Because these structures are sometimes difficult to distinguish in
histological section, we will refer to them collectively as nascent nephrons.
We found that Fgf8 RNA became restricted to a subset of cells within
the epithelium as the nascent nephrons matured
(Fig. 1E″), and to the
tubule progenitors in S-shaped bodies; Fgf8 RNA appeared to be absent
from future podocytes and Bowman's capsule cells
(Fig. 1E′′′).
As nephrogenesis proceeded, Fgf8 expression appeared to be
downregulated, as evidenced by the absence of a hybridization signal in
cortical regions containing nephrons that had matured past the S-shaped body
stage (Fig. 1E).

Expression of Fgf8 in the developing kidney and phenotype of
Fgf8-MM-KO kidneys at E18.5. (A) Schematic diagrams illustrating the
early stages of nephron formation (nephrogenesis; left) and the mature nephron
(right). During development, nephron and stroma progenitors are localized in
the peripheral zone near the outermost edge of the kidney primordium, whereas
the branching collecting duct system is localized to the central region of the
rudiment. Nascent nephrons form adjacent to collecting duct tips and develop
into S-shaped bodies that differentiate into the various cell types of the
nephron. During nephron maturation, invasion of the podocyte layer by
endothelial cells (not illustrated) leads ultimately to the formation of a
capillary tuft that becomes enveloped by podocytes. Together with the
surrounding squamous epithelium of Bowman's capsule, they comprise the renal
corpuscle portion of the mature nephron. The tubular portion of the nephron
consists of three distinct segments: the proximal tubule, the loop of Henle,
and the distal tubule, which connects to the collecting duct system. (B-E)
Whole mounts of E11.5-E12.5 mouse kidney primordia (B-D), and cryo-sections of
E16.5 kidneys (E), processed for in situ hybridization to localize expression
of Fgf8. At higher magnification, Fgf8 expression is
detected in (E′,E″) nascent nephrons and (E′′′)
the tubule progenitor cells within S-shaped bodies. (F) Analysis of
lacZ expression (blue staining) in sections through E12.5 kidneys
from embryos carrying the Pax3-cre transgene and the R26R
reporter allele. Note that only ureteric bud-derived collecting duct epithelia
do not express lacZ (pink staining). (G) Comparison of intact normal
and Fgf8-MM-KO kidneys at E18.5. (H,I) Immunofluorescence assays in
vibratome sections through E18.5 normal and Fgf8-MM-KO kidneys for
PAX2 (green), which normally labels nephron progenitors in the cortex as well
as collecting ducts present in both cortex and medulla, and smooth muscle
actin (SMA, red), which normally labels the stroma in the medulla, the
vasculature in the cortex, and the ureter. (J-K″) Low (J,K) and high
(J′,K′,K″) power views of cryo-sections, showing expression
of Wnt4 and Fgfr1 in E16.5 kidneys. Wnt4 and
Fgfr1 transcripts are detected in nascent nephrons. In addition,
Fgfr1 transcripts are detected in the peripheral zone and throughout
the S-shaped bodies. BC, Bowman's capsule progenitors; CD, collecting duct
progenitors; Co, cortex; Me(I), inner medullary region; Me(O), outer medullary
region; NN, nascent nephron; Po, podocyte progenitors; PZ, peripheral zone;
Tu, tubule progenitors; UB, ureteric bud; Ur, ureter.

Inactivation of Fgf8 in the metanephric mesenchyme

Because Fgf8 null embryos die before kidney formation initiates
(Sun et al., 1999), we
employed a Cre-mediated tissue-specific knock-out strategy to study the
consequences of loss of Fgf8 function in the metanephric mesenchyme.
To identify an appropriate source of Cre activity, we tested a
Pax3-cre transgene (Li
et al., 2000) that has been reported to function in the newborn
kidney (Chang et al., 2004), as
well as in the neural crest (Li et al.,
2000), for Cre activity at early stages of kidney development. In
embryos carrying both Pax3-cre and R26R, a reporter allele
in which lacZ is expressed only after Cre-mediated recombination
(Soriano, 1999), reporter gene
expression was detected at E11.5 and E12.5, in what appeared to be all
metanephric mesenchyme cells (Fig.
1F, and data not shown). A similar analysis using the Z/eG
reporter, which expresses lacZ before but not after Cre-mediated
recombination (Novak et al.,
2000), revealed that very few lacZ-expressing cells were
detected in the metanephric mesenchyme of Pax3-cre;Z/eG
double transgenic embryos (not shown). Together, these data show that
Pax3-cre activity leads to the virtually complete
recombination of floxed reporter alleles in the metanephric mesenchyme, and
thus should be useful for eliminating Fgf8 function in the developing
kidney by the stage when expression of Fgf8 is initiated (see
Fig. 1B,C).

To assess the efficiency at which Pax3-cre activity
deletes Fgf8 coding sequences during kidney development,
Pax3-cre;Fgf8null/+ males were crossed
to females homozygous for Fgf8flox, an Fgf8
conditional allele with wild-type activity that is converted by Cre-mediated
recombination to an Fgf8 null allele lacking exons 2 and 3
(Fgf8Δ2,3)
(Meyers et al., 1998). cDNA
prepared from extracts of kidney primordia isolated from their offspring at
E11.5 and E12.5, was used to perform quantitative PCR for the exon 2/3
sequences that are flanked by loxP sites in the
Fgf8flox allele. An Fgf8 exon 2/3 amplification
product was detected in all samples prepared from control (normal) embryos
that were either Fgf8flox/null or carried one copy of
Fgf8+ (n=6 at E11.5, n=4 at E12.5), but
no exon 2/3 amplification product was detected in samples prepared from the
Pax3-cre; Fgf8flox/null embryonic
kidneys (n=4 at E11.5, n=7 at E12.5; data not shown). These
results demonstrate that Pax3-cre activity results in a
complete loss of Fgf8 function in Pax3-cre;
Fgf8flox/null embryonic kidneys, although we cannot rule
out the possibility that there is a small amount of transient Fgf8
expression. Hereafter, we will refer to such embryos as Fgf8-MM-KO
(Metanephric Mesenchyme-Knock Out) mutants. Although born at the expected
Mendelian frequency, Fgf8-MM-KO mutants died shortly after birth.

At E18.5, all Fgf8-MM-KO kidneys were severely malformed
(Fig. 1G). At that stage,
normal kidneys comprise an inner medullary region, an outer medullary region
and a cortical region (Fig.
1H), each containing part of the PAX2-expressing collecting duct
system. The inner medullary region consists of mostly collecting ducts,
whereas the outer medullary region also contains abundant smooth muscle actin
(SMA)-expressing stroma, as well as the loop of Henle segment of the nephron.
The cortical region contains all other domains of the nephron (see
Fig. 1A). By contrast,
Fgf8-MM-KO kidneys were composed solely of what appeared to be an
inner medullary region containing very short PAX2-positive collecting ducts,
but lacked SMA-positive outer medullary stroma and all portions of the nephron
(Fig. 1I, and data not shown).
The ureters of Fgf8-MM-KO and normal kidneys were indistinguishable,
as determined by the presence of a well-developed smooth muscle coat and
terminally differentiated urothelium (not shown). Collectively these data
indicate that FGF8 signaling is essential for normal kidney development,
although the earliest events in kidney morphogenesis, including ureteric bud
formation and at least some collecting duct branching, occur in its
absence.

Nephrogenesis is initiated in Fgf8-MM-KO kidneys but does
not progress to the S-shaped body stage

To determine the cause of these abnormalities, we analyzed
Fgf8-MM-KO kidneys from the earliest stages of development. At E12.5,
the mutant kidneys appeared to be grossly normal (data not shown), but at
E13.0 and E14.5, they were markedly smaller than kidneys from their normal
littermates (Fig. 2). Despite
this size reduction, the patterns of expression of genes thought to mark
nephron progenitors in the peripheral zone, including Wt1, a
transcriptional regulator required at early stages of kidney development
(Kreidberg et al., 1993;
Donovan et al., 1999)
(Fig. 2A,B), Gdnf, a
signaling molecule essential for ureteric bud formation and branching
(Moore et al., 1996;
Pichel et al., 1996;
Sanchez et al., 1996)
(Fig. 2C,D) and PAX2, a
transcription factor required for Gdnf expression
(Brophy et al., 2001)
(Fig. 2Q,R), appeared to be
normal in Fgf8-MM-KO kidneys at E13.5 to E14.5. Likewise, the
expression of Foxd1 (formerly known as Bf2), which marks
stroma precursor cells (Hatini et al.,
1996) appeared normal at E14.5
(Fig. 2E,F). Collecting ducts,
marked by staining for Calbindin (CB) (Liu
et al., 1993), were also present
(Fig. 2Q,R), but the number of
collecting duct tips was reduced in the Fgf8-MM-KO kidneys to∼
45% of the normal number at E13.5 (see Table S2 in the supplementary
material), suggesting that by that stage collecting duct tips had on average
undergone one less branching event.

Nephrogenesis was initiated in Fgf8-MM-KO kidneys, as evidenced by
the presence of Wt1- (Fig.
2A,B) and PAX2- (Fig.
2Q,R) positive nascent nephrons, at least some of which appeared
to have reached the renal vesicle stage
(Fig. 2B,R), and also by the
expression of Fgf8, a marker for cells at early stages of
nephrogenesis (see Fig. 1E).
The latter was detected using a full-length Fgf8 probe that includes
sequences present in Fgf8null transcripts
(Meyers et al., 1998)
(Fig. 2G,H). These nascent
nephrons normally co-express Fgf8
(Fig. 1E′) and
Wnt4 (Stark et al.,
1994) (Fig.
1J′). However, Wnt4 RNA was not detected in
Fgf8-MM-KO kidneys at E13.5 (n=9) or E14.5 (n=3)
(Fig. 2I,J and data not shown).
Likewise, Lim1 expression was not detected in Fgf8-MM-KO
nascent nephrons at E13.5 (n=2) or E14.5 (n=3;
Fig. 2K,L). Consistent with the
lack of Wnt4 and Lim1 expression, both of which are required
for progression through the early stages of nephrogenesis
(Stark et al., 1994;
Kispert et al., 1998;
Kobayashi et al., 2005), few
if any S-shaped bodies were observed in the Fgf8 mutant kidneys at
any of the stages examined. It is likely that FGFR1 transduces the FGF8 signal
required for Wnt4 and Lim1 expression, as Fgfr1,
but not other FGF receptor family members, is abundantly expressed in nascent
nephrons (Stark et al., 1991;
Peters et al., 1992;
Peters et al., 1993;
Chi et al., 2004)
(Fig. 1K,K′, and Fig. S1
in the supplementary material). Together, our data show that, from early
stages, Fgf8-MM-KO kidneys are smaller than normal, but all
progenitor cell types thought to be essential for further development are
present, including nephron and stroma progenitors. Furthermore, nephrogenesis
initiates in the mutant kidneys, but rarely, if ever, progresses to the
S-shaped body stage.

Nephrogenesis arrests and cells in the peripheral zone die in the absence
of FGF8. Marker analysis in (A,B,Q-T) cryosections or (C-P,U,V) vibratome
sections of kidneys at the stages indicated. (A-L,U,V) In situ hybridization
for the genes indicated. The Fgf8 (FL) probe we used contained the
full-length coding sequence, and therefore detected Fgf8 RNA produced
by the Fgf8null allele. (M-P) Immunofluorescence assays
for PAX2 (green) to identify the developing nephrons and collecting ducts,
co-stained with LysoTracker (LysoT, red) to identify regions containing dying
cells. (Q-T) Immunofluorescence assays for PAX2 (green) and Calbindin (CB,
blue), which identifies collecting ducts, and for TUNEL staining (red), which
detects dying cells. Arrowheads point to nascent nephrons, which are present
in normal kidneys and also in Fgf8-MM-KO kidneys at E13.5 (A,B) and
E14.5 (Q,R), but not at E16.5 (S,T). Note that the nascent nephrons in
Fgf8-MM-KO kidneys (B,R) have formed an epithelial structure
surrounding a lumen, i.e. they have reached the renal vesicle stage.

As Wnt4 function appears to be required for Lim1
expression (Kobayashi et al.,
2005), we performed an experiment to determine whether
nephrogenesis in Fgf8-MM-KO kidneys could be rescued by providing a
source of WNT signaling. We therefore co-cultured metanephric mesenchyme
isolated from Fgf8-MM-KO kidney primordia with dorsal spinal cord, a
tissue that has previously been shown to rescue nephrogenesis in metanephric
mesenchyme isolated from Wnt4 null kidneys
(Kispert et al., 1998).
Staining for E-Cadherin (E-CAD), which in these cultures marks nephron
epithelia (Cho et al., 1998),
showed that after two days of co-culture with dorsal spinal cord, metanephric
mesenchyme isolated from control littermates contained numerous intensely
E-CAD-positive vesicular structures (n=10;
Fig. 3A). By contrast, under
the same culture conditions, metanephric mesenchyme isolated from
Fgf8-MM-KO embryos contained only weakly E-CAD-positive cell
aggregates (n=7; Fig.
3B), presumably representing the cells we detected in
Fgf8-MM-KO kidneys that have initiated nephrogenesis but then fail to
develop into S-shaped bodies. Significantly, no E-CAD-positive cells were
detected when metanephric mesenchyme was isolated from wild-type embryos and
cultured without dorsal spinal cord (see Fig. S2 in the supplementary
material). These data provide evidence that a source of signals that can
rescue nephrogenesis in Wnt4 null mesenchyme does not rescue this
process in Fgf8 null mesenchyme, suggesting that the lack of
nephrogenesis observed in the absence of FGF8 signaling is not due solely to a
lack of WNT4 signaling.

Cells in the peripheral zone die at an early stage in
Fgf8-MM-KO kidneys

In view of the small size of the Fgf8-MM-KO kidneys at early
stages (Fig. 2), we explored
the possibility that cell death contributes to this phenotype. In contrast to
normal kidneys at E14.5, which displayed sporadic cell death mostly in the
region where nephrons are forming (Koseki
et al., 1992; Coles et al.,
1993) (Fig. 2O,Q),
Fgf8-MM-KO kidneys at this stage contained a large number of cells
immediately underlying the capsule with the characteristics of apoptotic
cells, as determined by staining for LysoTracker
(Fig. 2P) and by TUNEL
(Fig. 2R) assays. Abnormal cell
death in the peripheral zone, although less widespread, was already observed
in the mutant kidneys at ∼E13.0 (Fig.
2M,N). Consistent with the extensive cell death observed at E14.5,
we found that by E16.5, PAX2-positive and Gdnf-expressing peripheral
cells were no longer present in Fgf8-MM-KO kidneys
(Fig. 2S,T and data not shown).
Significantly, numerous Foxd1-expressing cells were still detected in
the peripheral zone of the mutant kidneys at E16.5
(Fig. 2U,V), indicating that
the stroma progenitor cell population persists for longer than the nephron
progenitor cell population in the absence of FGF8. These results strongly
suggest that Fgf8 is required for the survival of nephron progenitor
cells in the peripheral zone, in which Fgfr1, but not other FGFR
genes, is abundantly expressed (Stark et
al., 1991; Peters et al.,
1992; Peters et al.,
1993; Chi et al.,
2004) (Fig.
1K-K″, see also Fig. S1 in the supplementary material).

Signals from the dorsal spinal cord are not sufficient to induce
nephrogenesis in Fgf8-deficient metanephric mesenchyme. (A,B)
Metanephric mesenchyme (MM) was isolated from E11.5 (A)
Fgf8flox/null (control) or (B)
Pax3-cre;Fgf8flox/null
(Fgf8-MM-KO) littermates and cultured in the presence of dorsal
spinal cord. After 48 hours of culture, the samples were processed for
immunohistochemistry to detect phospho-Histone H3, which marks cells in
mitosis (blue) and E-Cadherin, which marks epithelia (red). The arrowhead in A
indicates a region where the tubular nature of the E-CAD-positive structures
is particularly evident.

Tubular nephron segments are truncated in Fgf8 hypomorphic
kidneys

Because the complete loss of Fgf8 function during kidney
development results in a failure of nephron formation prior to the S-shaped
body stage, we were unable to determine whether FGF8 signaling is essential
for subsequent steps in nephrogenesis using Fgf8-MM-KO mutants.
Therefore, we analyzed the renal phenotype of mouse embryos in which
functional Fgf8 RNA is expressed at lower than normal levels in all
tissues throughout embryonic development. Embryos that are homozygous for
Fgf8neo, a hypomorphic allele
(Fgf8neo/neo embryos; referred to as mild Fgf8
hypomorphs), or compound heterozygotes for Fgf8neo and an
Fgf8 null allele (Fgf8neo/null embryos; referred
to as severe Fgf8 hypomorphs), have been roughly estimated to produce∼
40% and ∼20% of wild-type levels of Fgf8 RNA, respectively
(Meyers et al., 1998). At
E18.5, the kidneys of these mutants were markedly smaller than control
kidneys, but were larger than Fgf8-MM-KO kidneys (compare
Fig. 4A and
Fig. 1G).

Interestingly, we detected Wnt4-expression in nascent nephrons in
E15.5 kidneys from severe Fgf8 hypomorphs
(Fig. 4B,C), demonstrating that
even the relatively low level of functional Fgf8 expression in these
mutants is sufficient to induce and/or maintain some Wnt4 expression.
Nephrogenesis was able to progress in these mutants, as shown by the presence
of renal corpuscles containing PECAM-positive capillary tufts
(Baldwin et al., 1994) and
WT1-positive podocytes (Armstrong et al.,
1993) at E15.5 (Fig.
4D,D′). Furthermore, podocytes were identified in E18.5 mild
and severe Fgf8 hypomorph kidneys by in situ hybridization assays for
Podxl (Doyonnas et al.,
2001) (Fig. 5A-C).
However, morphometric analyses performed at E15.5 and E18.5 showed that the
number of renal corpuscles was reduced in mild (from ∼70% to ∼35% of
normal), and even more reduced in severe (from ∼50% to ∼20% of
normal), Fgf8 hypomorph kidneys (see Table S3 in the supplementary
material). This reduction can presumably be explained, at least partially, by
a reduction in collecting duct system branching (see Table S2 in the
supplementary material).

The presence of renal corpuscles in kidneys from Fgf8 hypomorphs
suggested that the tubular segment of the nephron might also be present. To
address this possibility, we performed an in situ hybridization analysis at
E18.5, using markers that identify various regions of maturing nephron tubules
(see Fig. 1A):
Slc34a1, which encodes a Na/Pi co-transporter expressed in
the proximal tubular segment (Murer et
al., 2004); Uromodulin, which encodes the Tamm-Horsfall protein
produced in the loop of Henle (Bachmann et
al., 1990); and Slc12a3, which encodes a Na/Cl
co-transporter expressed in the distal tubular segment
(Hebert et al., 2004). This
analysis showed that proximal convoluted tubules and distal tubules were
substantially reduced in mild hypomorphs and barely detectable in severe
hypomorphs, whereas the loop of Henle was not present in either mutant
(Fig. 5D-L).

To determine whether this reduction or lack of gene expression was caused
by a failure of nephron segments to express these differentiated tubule
segment markers or whether it was a reflection of the absence of the tubule
segments, we performed an immunofluorescence assay to detect nephron tubules.
Because there is no single antibody that labels only nephron tubules, we
double stained with two antibodies: anti-E-CAD, which labels both nephron
tubules and collecting ducts (red) (Cho et
al., 1998); and anti-CB, which labels collecting ducts only
(green). Thus, the nephron tubules that stain for E-CAD only (red) can be
distinguished from the collecting ducts that stain for both E-CAD and CB
(yellow) (Fig. 6A). This
analysis showed that at E18.5, the nephron tubules were indeed virtually
absent in kidneys from severe Fgf8 hypomorphs
(Fig. 6C). Furthermore, an
inspection of nephrons isolated from severe hypomorphs showed that the renal
corpuscles were connected to the collecting system by a severely truncated
tubular segment (Fig. 6B,D).
These data demonstrate that low levels of FGF8 signaling support the formation
of a limited number of nephrons with severely truncated tubular segments.

We also found that wild-type kidney rudiments cultured in the presence of
function-blocking antibodies against FGF8 had much shorter tubule segments
than were observed in control cultures
(Fig. 6E-F′),
phenocopying the Fgf8 hypomorph nephron tubule phenotype. These data
demonstrate that Fgf8 is required within the developing kidney to
support full tubule development, and argue against the possibility that the
Fgf8 hypomorph phenotype is secondary to an early defect in the
development of the intermediate mesoderm, caused by reduced Fgf8
expression during gastrulation in the hypomorphs.

Fgf8 is required for cell survival within developing
nephrons

To identify the cause of nephron tubule truncation, we focused our
attention on the more severely affected Fgf8neo/null
kidneys. Assays for cell death at E14.5
(Fig. 7A-B′) revealed
that, as in normal kidneys, some dying cells were observed in the developing
renal corpuscle in severe Fgf8 hypomorphs (arrows in
Fig. 7A′,B′).
However, in addition, there were numerous dying cells in the tubule
progenitors in the S-shaped bodies of the mutant kidneys (arrowhead in
Fig. 7B′), in which
Fgfr1, but not other FGFR genes, is abundantly expressed
(Stark et al., 1991;
Peters et al., 1992;
Peters et al., 1993;
Chi et al., 2004)
(Fig. 1K,K″, and Fig. S1
in the supplementary material). We did not detect the extensive peripheral
cell death that was observed in Fgf8 MM-KO kidneys at this stage
(compare Fig. 7B with
Fig. 2P). These data suggest
that the reduced tubular length observed in E18.5 hypomorphic kidneys is due
to the death of tubule progenitors at the S-shaped body stage. At E15.5, the
Fgf8neo/null kidneys continued to display abnormal cell
death in S-shaped bodies, but, in addition, cell death was also observed in
the peripheral zone (Fig.
7C-D′). These data indicate that Fgf8 plays a role
in sustaining the survival of the tubule progenitors present in S-shaped
bodies at early stages of nephrogenesis, as well as of progenitor cells in the
peripheral zone of the developing kidney.

Nephrons are truncated in severe Fgf8 hypomorphs and in cultures
of wild-type kidney explants treated with anti-FGF8 antibody. (A,C)
Immunofluorescence assays in vibratome sections of E18.5 kidneys from normal
and severe Fgf8 hypomorphs. Podocytes are marked by staining for WT1
(blue). Collecting ducts are marked by staining for both E-CAD (red) and
Calbindin (green), and thus appear yellow. The tubular portion of the nephron
is marked by staining for E-CAD only. Note that the cortex of the hypomorphic
kidney lacks the abundant, red tubular structures present in the normal
kidney. (B,D) Representative nephrons isolated from E18.5 kidneys better
reveal the lengths of the tubules. (E,F) Immunofluorescence assays for E-CAD
(red) to identify epithelia, and WT1 (green) to identify podocyte progenitors
of (E) control and (F) anti-FGF8 antibody-treated cultures of wild-type kidney
explants. (E′,F′) Higher magnification views. The black arrowheads
outlined in yellow indicate the proximal end of a tubule, where it is
connected to the renal corpuscle. The white arrowheads indicate the nearest
branch point of the collecting duct to which the tubule is connected. Note the
decrease in tubule length in the nephrons from the Fgf8 severe
hypomorph kidney and the anti-FGF8 antibody-treated cultures.

Discussion

Using an allelic series of Fgf8 mutants, we have investigated the
role of FGF8 in kidney development. We found that complete inactivation of
Fgf8 in the metanephric mesenchyme results in the formation of
kidneys that are severely malformed at birth, lacking both the outer medullary
and cortical regions, and thus are devoid of nephrons and most of the
collecting duct system. Our analysis of Fgf8-MM-KO kidneys at early
stages of development indicated that this final phenotype is the consequence
of two defects: a failure of nascent nephrons to progress to the S-shaped body
stage and death of nephron progenitors in the peripheral zone. These defects
are attributable, at least in part, to a lack of Wnt4 and
Lim1 expression in the absence of FGF8. Using a different
cre transgene to eliminate Fgf8 function, Perantoni et al.
(Perantoni et al., 2005) came
to similar conclusions about FGF8 function in kidney development. In
hypomorphic mutants, Fgf8 expression is severely reduced but
sufficient to allow nephrogenesis to proceed. However, cells in the tubular
portion of the developing nephrons die, resulting in severe truncation of the
nephrons. Thus, analysis of Fgf8 null and hypomorphic embryonic
kidneys in which FGF8 signaling is either absent or severely reduced has
enabled us to demonstrate roles for FGF8 in the regulation of gene expression
and in cell survival essential for nephron formation.

Cells within S-shaped bodies die in Fgf8 hypomorphs. (A-D)
Immunofluorescence assay for PAX2 (green), to identify the developing nephrons
and collecting ducts, and LysoTracker (LysoT, red) staining to identify
regions containing dying cells in vibratome sections of normal and
Fgf8 severe hypomorph kidneys at the stages indicated.
(A′-D′) Higher magnification views. Arrows indicate regions where
Bowman's capsule progenitors are dying in normal and mutant kidneys; white
arrowhead indicates regions where tubule progenitors are dying in the mutant
kidney; open arrowheads point to regions in the peripheral zone of the mutant
kidney where cells are dying.

Fgf8 is required at early stages of nephrogenesis

Nephron formation is initiated in the absence of FGF8, as evidenced by the
finding that renal vesicles, marked by Wt1, PAX2 and non-functional
Fgf8 expression, are present at E13.5 and E14.5. However, we detected
neither Wnt4 nor Lim1 RNA, suggesting that FGF8 is required
to induce and/or maintain expression of these genes. Alternatively, FGF8 may
affect Wnt4 and Lim1 expression less directly; for example,
by ensuring that cells in the nascent nephrons are competent to express these
genes. In either case, the data suggest a model whereby FGF8 produced in the
nascent nephron acts in an autocrine fashion in a genetic pathway(s) upstream
of Wnt4 and Lim1. Consistent with this model, nascent
nephrons in kidneys that lack Wnt4
(Perantoni et al., 2005) or
Lim1 (Kobayashi et al.,
2005) function still express Fgf8. Because Lim1
expression depends on Wnt4 function
(Kobayashi et al., 2005), the
lack of Lim1 expression and the consequent arrest of nephrogenesis in
Fgf8-MM-KO kidneys might be caused entirely by the lack of
Wnt4 expression. However, this seems unlikely because we found that a
source of WNT signals (dorsal spinal cord) did not rescue nephrogenesis in
cultures of Fgf8-MM-KO metanephric mesenchyme. Therefore, the effect
of FGF8 on Lim1 expression is not solely due to its effect on
Wnt4 expression. Instead, FGF8 may act in conjunction with WNT4 to
regulate Lim1 expression.

The lack of Lim1 expression readily explains the failure of the
nascent nephrons in Fgf8-MM-KO mutant kidneys to progress to the
S-shaped body stage, as the same phenotype is observed when Lim1 is
inactivated in the metanephric mesenchyme
(Kobayashi et al., 2005). What
is more puzzling is why renal vesicles are present in the apparent absence of
Wnt4 expression in Fgf8-MM-KO mutant kidneys, as
Wnt4 is thought to be required for nephron progenitors to undergo a
mesenchymal to epithelial transition, i.e. to form renal vesicles
(Stark et al., 1994;
Kispert et al., 1998). One
possible explanation is that Fgf8 function is not required to induce
Wnt4 expression, but only to upregulate/maintain it. If so, then
there may be transient Wnt4 expression in the Fgf8-MM-KO
kidneys that we did not detect, but which is sufficient to allow renal
vesicles to form. Alternatively, it may be that differences in genetic
background between the Wnt4 null and the Fgf8-MM-KO mutant
mice may influence the penetrance of the block to renal vesicle formation
caused by the absence of Wnt4. In this context, it is interesting to
note that a small number of S-shaped bodies are present in Wnt4 null
kidneys at E14.5 (Kobayashi et al.,
2005). This indicates that at least some nephron progenitors do
undergo a mesenchymal to epithelial transition in the Wnt4 mutants,
perhaps in response to other WNT signals. Limited nephrogenesis can
subsequently occur in Wnt4 null kidneys
(Kobayashi et al., 2005;
Perantoni et al., 2005),
presumably because FGF8 is still produced and available to induce
Lim1. By contrast, neither S-shaped bodies nor nephrons can form in
Fgf8-MM-KO kidneys because FGF8 is not available to induce
Lim1 expression.

Fgf8 is required for cell survival in the developing
kidney

By examining animals carrying the Fgf8neo hypomorphic
allele, in which sufficient FGF8 is produced to support the formation of
S-shaped bodies, we were able to uncover a function for FGF8 at later stages
of nephrogenesis. We found that a substantial number of tubule precursor cells
in the S-shaped bodies of Fgf8neo/null and
Fgf8neo/neo kidneys die
(Fig. 7, and data not shown).
We therefore conclude that FGF8 signaling is required within S-shaped bodies
for cell survival, thus providing an explanation for our finding that nephron
tubule length is shorter than normal in Fgf8neo/neo
kidneys, which produce less FGF8 than normal, and is dramatically reduced in
Fgf8neo/null kidneys, which produce even less FGF8.

FGF8 signaling is also required for the survival of cells in the peripheral
zone, as we detected abnormal cell death in Fgf8-MM-KO kidneys as
early as E12.5 (data not shown), presumably accounting for the reduced size of
the null mutant kidneys at early stages. At E14.5, a large number of cells in
this region are dying, although both PAX2- and Foxd1-expressing
peripheral cell populations are still present. However, by E16.5, the
PAX2-positive nephron progenitor cell population is absent, presumably because
it is eliminated by cell death at earlier stages. By contrast,
Foxd1-expressing stroma progenitors are still detected at E16.5.
However, even if they continue to remain viable, these cells never form
SMA-positive stroma because the entire outer medullary and cortical regions of
the mutant kidneys, where SMA-positive cells normally reside, fail to develop
in Fgf8-MM-KO kidneys. The progressive loss of nephron progenitors,
which produce the GDNF required for collecting duct branching, may also
account for the reduction in the number of collecting duct tips that we
observed in Fgf8-MM-KO kidneys at all stages analyzed (see Table S2
in the supplementary material). Thus all of the defects that we observed in
the neonatal Fgf8-MM-KO kidneys can be explained by the requirement
for FGF8, both for gene expression essential for nephrogenesis and for the
survival of nephron progenitors in the peripheral zone.

Because kidney development depends on reciprocal interactions between its
tissue components, it is difficult to determine whether a specific defect
observed in mutant kidneys reflects the primary function of the mutated gene.
This is especially true when the gene in question encodes a secreted protein
such as FGF8, which could act locally or at a distance to exert its effects.
Thus, it is unclear whether the death of nephron progenitors observed in
Fgf8-MM-KO kidneys is directly or indirectly due to the lack of FGF8.
If it is a direct effect, then the FGF8 signal produced in nascent nephrons is
probably transduced by FGFR1, the only FGF receptor gene abundantly expressed
in that zone (Stark et al.,
1991; Peters et al.,
1992; Peters et al.,
1993; Chi et al.,
2004) (Fig.
1K-K″; see also Fig. S1 in the supplementary material).
Moreover, the effect presumably occurs over a relatively long distance,
because at the stage when there is extensive cell death in the
Fgf8-MM-KO peripheral zone Fgf8 expression in normal embryos
appears to be restricted to the nascent nephrons.

In support of a direct effect, previous studies have shown that FGF
signaling can prevent cell death in the metanephric mesenchyme. Thus, addition
of FGF2 to metanephric mesenchyme isolated at E11.5 and cultured in the
absence of the ureteric bud prevents the cell death that is normally observed
in such cultures, although it does not induce nephrogenesis
(Perantoni et al., 1995;
Barasch et al., 1997;
Dudley et al., 1999). We have
found that FGF8 can likewise sustain cell survival and proliferation, but is
not sufficient to induce the formation of nascent nephrons in isolated
wild-type metanephric mesenchyme (see Fig. S2 in the supplementary material).
Because no kidney defect has been reported in Fgf2 null mice (Dono,
1998; Ortega et al., 1998),
whereas we show that FGF8 is required in vivo for survival of nephron
progenitor cells, it appears that the effects of FGF2 in vitro may in fact be
mimicking the survival function of FGF8 in normal kidney development.

Another secreted factor, BMP7, is capable of supporting the survival of
isolated metanephric mesenchyme without inducing nephrogenesis, and BMP7 has
been shown to act synergistically with FGF2 in that assay
(Dudley et al., 1999). During
kidney development, Bmp7 is normally expressed in the peripheral
zone, in the developing nephron at early stages, and also in the branching
collecting ducts (Godin et al.,
1998). Although the phenotype of Bmp7 null kidneys
appears to be less severe than that of Fgf8-MM-KO kidneys
(Dudley et al., 1995;
Luo et al., 1995),
Bmp7 null and Fgf8-MM-KO kidneys both display abnormal cell
death in the peripheral zone starting at E12.5-E13.5, and by E16.5 the nephron
progenitors in the peripheral zone are eliminated
(Luo et al., 1995;
Dudley and Robertson, 1997).
These observations raise the possibility that, in vivo, BMP7 and FGF8
cooperate to maintain progenitor cell populations in the peripheral zone.

Alternatively, cell death in the peripheral zone caused by lack of
Fgf8 function may be due to the observed absence of Wnt4
expression in nascent nephrons. Support for this suggestion comes from a
recent observation that there is extensive death in the peripheral zone in
Wnt4 mutant kidneys at E13.5 and E14.5 (S. Vainio and P.
Itäranta, personal communication), similar to that observed in
Fgf8-MM-KO kidneys. These findings are consistent with the
possibility that the role of FGF8 in cell survival in the peripheral zone is
to induce and/or maintain the expression of Wnt4 in nascent nephrons,
and that WNT4 alone, or acting in conjunction with FGF8 or some other factor,
promotes the survival of nephron progenitors.

Concluding remarks

The concept that FGF8 is required for cell survival during organogenesis
has been a recurrent theme in studies on Fgf8 function in
development. For instance, loss of Fgf8 function in developing limb
buds (Sun et al., 2002;
Boulet et al., 2004), first
branchial arch (Trumpp et al.,
1999), forebrain (Storm et
al., 2003), midbrain/anterior hindbrain
(Chi et al., 2003), and, as
shown here, developing kidneys, results in extensive cell death. In some of
these cases, the regions in which dying cells are found in the absence of FGF8
signaling are distant from the site of FGF8 synthesis. A challenge for the
future will be to determine precisely how FGF signaling functions to promote
cell survival in these different developmental settings.

Acknowledgments

We thank Drs. J. Epstein and P. Soriano for kindly providing the
Pax3-cre and R26R mouse lines. We are grateful to C. Ahn and
P. Ghatpande for excellent technical assistance. We also thank T. Carroll, M.
Lewandoski, A. McMahon, and S. Vainio for sharing unpublished data and helpful
discussion, and to our laboratory colleagues for critical reading of the
manuscript. C.C. was supported by a postdoctoral fellowship from Ministerio
Español de Educación Cultura y Deporte. This work was supported
by NIH PO1 HD39948 (E.N.M.) and RO1 grants HD42803 (E.N.M.), DK45218 and
DK56365 (D.H.) and HD34380 (G.R.M.).

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